Microfluidic device, system and methods thereof for measuring and recording electrical signals from a pool of multiple nematodes

ABSTRACT

The present disclosure provides a microfluidic device and system for measuring a composite electropharyngeogram (EPG) signal from a pool of multiple nematodes, wherein the composite EPG signal is measured from the pool of nematodes present in a single recording channel connected to two or more integrated electrodes. The microfluidic device includes an inlet port and outlet port directly connected to a single recording channel and two or more electrodes directly connected to the recording channel. The recording channel is configured to hold 10 to 10,000 nematodes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/689,052, filed on 22 Jun. 2018, the content of whichis incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under MH051383 awardedby National Institutes of Health. The government has certain rights inthe invention

FIELD OF THE INVENTION

This application pertains generally to microfluidic devices and methodsthereof for measuring and recording composite electropharyngeogram (EPG)signals from a pool of multiple nematodes.

BACKGROUND OF THE INVENTION

Parasitic nematodes are major contributors to disease in humans,livestock, and companion animals. In addition, the non-parasiticnematode Caenorhabditis elegans is both a major model organism for basicresearch in biology and medicine, and a validated screening organism fordeveloping drugs and nematicides to control parasitic species.

The two main measures of nematode health and dysfunction involvequantification of: (i) movements related to locomotion and (ii) musclecontractions associated with feeding. For adequate statisticalsignificance, both measures often require the assessment of tens tohundreds of nematodes per experimental condition, such as when testingdifferent types of drugs. Numerous methods exist for simultaneousquantification of locomotion in hundreds of individual nematodes, forexample by tracking them in videos as they crawl across a laboratorysubstrate. Itskovits E, Levine A, Cohen E, Zaslaver A. A multi-animaltracker for studying complex behaviors. BMC Biol. 2017 Apr. 6; 15(1):29;Swierczek N A, Giles A C, Rankin C H. High-throughput behavioralanalysis on C. elegans. Kerr R A. Nat Methods. 2011 Jun. 5; 8(7):592-8.However, the only methods for simultaneous quantification of feeding innematode populations involve either the measurement of food consumptionor the accumulation of fluorescently labeled food in the animal. Bothapproaches are indirect, and neither is capable of quantifyingindividual swallowing events.

The nematode feeding organ is the pharynx, a rhythmically activemuscular pump that sucks nutrients from the environment and passes theminto the gut for digestion. Each pharyngeal contraction, called a pump,generates an electrical event that can be recorded by electrodes inelectrical contact with the nematode's body. Such a recording is calledan electropharyngeogram (EPG). It has been shown that EPGs can berecorded from a single nematode by placing it in tight fittingmicrofluidic channel filled with a conductive buffer solution that is incontact with electrodes within the channel. See U.S. Pat. No. 9,723,817,herein incorporated by reference. That device, however, only records EPGmeasurements from individual nematodes, accommodating up to eightnematodes per microfluidic device, and does not provide a high throughput means for measuring and recording EPG signals from a largepopulation of nematodes. Lockery S R, Hulme S E, Roberts W M, Robinson KJ, Laromaine A, Lindsay T H, Whitesides G M, Weeks J C. A microfluidicdevice for whole-animal drug screening using electrophysiologicalmeasures in the nematode C. elegans. Lab Chip. 2012 Jun. 21;12(12):2211-20.

Others have described alternative methods for simultaneousquantification of feeding in nematode populations. One such method isthe bacteria clearance assay, as in Gomez-Amaro R L et al., MeasuringFood Intake and Nutrient Absorption in Caenorhabditis elegans. Genetics.2015 June; 200(2):443-54. In that approach, 3-14 nematodes are loadedinto micro wells containing a liquid suspension of bacteria. As thenematodes feed, bacteria are removed from the liquid causing a reductionin its optical density. Feeding is quantified as the change in opticaldensity between two time points, normalized to the number of nematodesin the well. Key drawbacks of the bacteria clearance assay include thefact that: i) for accurate normalization, nematodes must be countedaccurately as they are added to the microwells, requiring expensiveinstrumentation or manual loading, ii) the assay is limited to 14 orfewer nematodes per well, a limitation that arises because when thenumber exceeds 14, changes in optical density per unit time are nolonger a linear function of the number of nematodes in the well, andiii) the assay does not measure feeding movements directly.

Moreover, the reliability of the bacteria clearance assay can becompromised by run-to-run variations in the quality of the bacteriapreparations, including such properties as bacterial growth phase, whichcan alter food consumption rates even when pumping rates are unaffected.This discrepancy occurs because in the logarithmic growth phase manybacteria are in a dividing state, making them larger and thus harder toswallow (Gomez-Amaro et al. 2015).

A second method described by others and used to approximate simultaneousquantification of feeding in nematode populations is a fluorescenceaccumulation method. Boyd et al. Effects of genetic mutations andchemical exposures on Caenorhabditis elegans feeding: evaluation of anovel, high-throughput screening assay. PLoS One. 2007 Dec. 5; 2(12). Inthat method, a population of nematodes is fed a mixture of E. colibacteria and fluorescent microspheres for 15 min. After feeding,nematodes are paralyzed so they can no longer swallow food and the totalaccumulated fluorescence of each nematode f_(T) is measured by passingeach nematode through a COPAS Biosort, a flow cytometer adapted for useon nematodes. Feeding is quantified for each nematode as f_(T)/t, wheret is the time-of-flight of that nematode in the flow cytometer, anapproximate measure of nematode length and thus overall size. Use of aflow cytometer can be expensive, e.g. $400,000/instrument or more, andthe assay is both an indirect measure of feeding rate and limited toquantifying for a 15-minute feeding period.

Accordingly, there is a need for a device and methods thatsimultaneously quantifies direct feeding events from a large number ofnematodes and that does not require prohibitively expensive equipment toperform.

The instant system provides a direct rather than indirect measure of thefeeding rate and provides advantages as compared to known methods anddevices. The advantages include: i) quantification of feeding periodsthat exceed 15 minutes (fluorescence accumulation method limitation);ii) no required knowledge of the number of nematodes in the device(bacteria clearance assay limitation); and, iii) no run to runvariability due to variations in bacterial food (bacteria clearanceassay limitation).

Provided herein is a microfluidic device, wherein the length and widthof the channel are increased to accommodate a population of nematodes,yielding a composite EPG from a pool of multiple nematodes which is thesum of the EPGs from each nematode in the channel. The advantage overknown microfluidic devices for recording EPG signals is the ability torecord the composite EPG signal from a large number of nematodes in asingle EPG recording channel. Recording EPG signals in single-nematodeEPG channels critically depends on the seal resistance which formsbetween the nematode and the channel. We herein demonstrate that thepresent microfluidic device provides a sufficient seal resistance toobtain useable recordings when the channel is enlarged to accommodate alarge population of nematodes.

The device therefore has the potential to accelerate nematode research,including drug and toxicology screening, by vastly increasing thethroughput of EPG methodologies.

SUMMARY OF THE INVENTION

Herein are provided devices, systems and methods for measuring acomposite electropharyngeogram (EPG) signal from a pool of multiplenematodes. In embodiments, the microfluidic device comprises an inletport and outlet port directly connected to a holding reservoir, a singlerecording channel connected in series to the holding reservoir and, twoor more integrated electrodes directly connected to the recordingchannel. In embodiments, the microfluidic system comprises an inlet portand outlet port directly connected to a holding reservoir, a singlerecording channel connected in series to the holding reservoir, two ormore integrated electrodes directly connected to the recording channel,and at least one differential amplifier or at least one voltage-clampamplifier, wherein the amplifier is connected to an output from the twoor more integrated electrodes.

In alternative embodiments, the microfluidic device comprises a) aninlet port and outlet port directly connected to a single recordingchannel; wherein the single recording channel, configured to hold 10 to10,000 nematodes; a tube placed in each of the inlet port and outletport, wherein the tube placed in the outlet port comprises a filter toretain nematodes in the single recording channel; and, two or moreelectrodes connected to the recording channel. In embodiments, the tubesare metal and function as the electrodes.

In embodiments, the recording channel is configured to hold 10 to 10,000nematodes. In certain embodiments, the recording channel is 10 mm to 500mm in length and 10 μm to 500 μm in width. In certain embodiments, therecording channel is 10 mm to 500 mm in length and 10 μm to 1mm inwidth. In embodiments, device comprises a silicone polymer, athermoplastic polymer, an acrylic polymer, or a polycarbonate polymer.In certain embodiments, the thermoplastic polymer comprises poly(methylmethacrylate) (PMMA), polycarbonate (PC), polystyrene (PS), polyvinylchloride (PVC), polyimide (PI), olefin polymers, cyclic olefin copolymer(COC), cyclic olefin polymer (COP), or cyclic block copolymer (CBC). Incertain other embodiments, the silicone polymer comprises apolydimethylsiloxane (PDMS) elastomer.

In embodiments, the pool of nematodes comprises Caenorhabditis elegans(C. elegans). In certain embodiments, the pool of nematodes comprisesparasitic nematodes. In certain other embodiments, the pool of nematodescomprises transgenic or variant nematodes. In embodiments, the nematodesexpress one or more human genes. In alternative embodiments, the pool ofnematodes comprises wild type nematodes.

Disclosed herein are methods for recording a compositeelectropharyngeogram (EPG) signal from a pool of multiple nematodes andmethods for screening test compounds by recording a compositeelectropharyngeogram (EPG) signal from a pool of multiple nematodes. Inembodiments, a method for recording a composite EPG signal comprisesintroducing the pool of multiple nematodes into the holding reservoirthrough the inlet port of the microfluidic system, wherein the pool ofmultiple nematodes is present in an aqueous buffer, moving the pool ofmultiple nematodes into the single recording channel, measuringelectrophysiological signals from the pool of multiple nematodes, andrecording the electrophysiological signals as a single composite EPG. Inembodiments, the buffer solution comprises serotonin, food, or otherstimulant to cause pharyngeal pumping.

In certain embodiments, the methods herein utilize the microfluidicdevice that does not comprise a holding reservoir, wherein the pool ofmultiple nematodes is introduced into the single recording channelthrough the inlet port of the microfluidic system, wherein the pool ofmultiple nematodes is present in an aqueous buffer solution, measuringelectrophysiological signals from the pool of multiple nematodes and,recording the electrophysiological signals as a single composite EPG.

In embodiments, a method for screening a test compound comprisescontacting a pool of multiple nematodes with the test compound,measuring and recording a composite EPG signal from the contacted poolof multiple nematodes, comparing the recorded composite EPG to a controlcomposite EPG, and determining if the recorded composite EPG is alteredas compared to the control composite EPG, whereby test compounds arescreened. In embodiments, determining if the recorded composite EPG isaltered comprises determining the power spectrum of the composite EPG,the frequency of the peak power of the composite EPG, the amplitude ofthe composite EPG, waveform of the composite EPG, or a combinationthereof.

In alternative embodiments, a method for screening a test compoundutilize the microfluidic device that does not comprise a holdingreservoir wherein tubes are placed in each of the inlet port and theoutlet port wherein at least the tube in the outlet port comprises amesh that maintains the nematodes in the recording channel whileallowing for exchange or flow of buffer through the recording channel.In embodiments, a method for screening a test compound comprises use ofa first buffer and a second buffer, wherein either of the first orsecond buffer comprises the test compound and the buffer can beexchanged without removing the pool of multiple nematodes from therecording channel.

In certain embodiments, a method for screening a test compound comprisescontacting the pool of multiple nematodes with a first buffer, measuringand recording a composite EPG from the first buffer pool of multiplenematodes, perfusing the pool of multiple nematodes with a second buffer(e.g., exchanging the first buffer for the second buffer), measuring andrecording a composite EPG from the second buffer pool of multiplenematodes, comparing the recorded composite EPG from the first bufferpool of multiple nematodes to the recorded composite EPG from the secondbuffer pool of multiple nematodes and, determining if the recordedcomposite EPG from the first buffer pool of multiple nematodes isaltered as compared to the recorded composite EPG from the second bufferpool of multiple nematodes, whereby test compounds are screened, whereineither of the first buffer or second buffer comprises a test compoundand the other buffer is a control buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like numerals describe similar components throughoutthe several views and different Figure numbers. The drawings illustrategenerally, by way of example, but not by way of limitation, variousembodiments disclosed herein.

FIG. 1 shows a top view and an end view of an exemplary microfluidicdevice 100. The microfluidic chip 105 includes an inlet port 125 andoutlet port 130, connected in parallel to a reservoir 115, that isconnected in series to a recording channel 120 wherein three electrodes135, 140 (ground electrode) and 145 are connected to the recordingchannel 120. The microfluidic chip 105 is secured to a backing 110 (e.g.glass plate or microscope slide) to form the microfluidic device 100.

FIG. 2 shows a top view of an exemplary microfluidic system 200 thatincludes the microfluidic device 100 placed in a recording dock 205 thatis connected to a differential amplifier 210.

FIGS. 3A, 3B and 3C show recordings of EPG measurements from individualnematodes; FIG. 3D shows an in silico composite EPG recording from 22individual nematode EPG measurements; and FIG. 3E shows a power spectrumfor the in silico composite EPG recording, using the microfluidic device100.

FIGS. 4A-4C show recordings of EPG measurements from a pool of multiplenematodes in M9 buffer with serotonin (FIG. 4A); M9 buffer withoutserotonin (FIG. 4B); and, a power spectrum of the EPG measurements froma pool of multiple nematodes (FIG. 4C), using the microfluidic device100.

FIG. 5A shows a top view of an exemplary microfluidic device 500. Themicrofluidic chip 505 includes an inlet port 520 and outlet port 525,connected to a recording channel 515. The microfluidic chip 505 issecured to a backing 510 (e.g. glass plate or microscope slide) to formthe microfluidic device 500. FIG. 5B shows the stainless-steel tubes 530and 535 in position to be inserted into the inlet port 520 and outletport 525. A nylon mesh 540 covers the opening of stainless-steel tube535 inside the microfluidic chip 505. FIG. 5C presents a side view ofthe microfluidic device 500 with the stainless-steel tube 535 inposition to be inserted into the outlet port 525 and the nylon mesh 540cover the opening of stainless-steel tube.

FIG. 6 shows a top view of an exemplary microfluidic system 600 thatincludes the microfluidic device 500 that is connected to a differentialamplifier 210, and amplifier cable 605 fitted with clips 610 to connectthe amplifier 210 to the microfluidic device 500.

FIG. 7A-7C shows recordings of EPG measurements from a pool of multiplenematodes in M9 buffer without serotonin (FIG. 7A); M9 buffer withserotonin (FIG. 7B); and, power spectra of the EPG measurements from thepool of multiple nematodes recorded in FIGS. 7A and 7B (FIG. 7C), usingthe microfluidic device 500.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Provided herein are microfluidic devices, systems, and methods of usethereof for measuring and recording a composite electropharyngeogram(EPG) from a pool of multiple nematodes. The instant microfluidicdevices disclosed herein measure and record electrical signals thatreveal the average frequency of individual swallowing eventssimultaneously in populations of nematodes. The present device, systemand method are an improvement over microfluidic devices designed tomeasure and record an EPG from individual nematodes wherein thosedevices, discussed in the background section, require the placement ofindividual nematodes into single-nematode EPG channels, each possessingits own electrodes and differential amplifier. The device and systemdisclosed herein utilize a pool of multiple nematodes placed in a singleEPG channel (e.g. recording channel).

Measuring and recording EPG signals in single-nematode EPG channelscritically depends on the seal resistance which forms between thenematode and the channel. Unexpectedly, and as demonstrated in Example 2and shown in FIG. 4 , expansion of a recording channel to accommodate apool of multiple nematodes (e.g., 10 or more) provided sufficient sealresistance to obtain useable recordings (e.g. composite EPGs).

In one embodiment, the microfluidic devices disclosed herein comprise aninlet port and outlet port connected in parallel to a holding reservoirthat is connected in series to a recording channel. See FIG. 1 . In thisembodiment, the pool of nematodes is loaded into the holding reservoirand then moved into the recording channel, such as by concentrating thenematodes via centrifugation.

In certain other embodiments, the microfluidic devices disclosed hereincomprise an inlet port and outlet port located at each end of therecording channel, wherein no holding reservoir is present in thisembodiment. The inlet and outlet ports further comprise a tube whereinthe end placed in the port comprises a mesh to block and maintain thenematodes in the recording channel. See FIG. 5 . In this embodiment, thepool of nematodes is loaded directly in the recording channel via theinlet port, wherein the nematodes remain trapped due to the mesh on theend of the tube in the outlet port. The mesh acts as a filter, making itpossible to create a high packing density of nematodes by continuing toinject nematodes until the recording channel is full.

In embodiments, the microfluidic device of FIG. 5 permits perfusion of atest compound during an experiment without removal of nematodes from therecording channel. In certain embodiments, recordings can be obtainedbefore and after perfusion with a test compound, such as a drugcandidate, in the same population of nematodes providing methods withinternal controls when screening test compounds.

Definitions

As used herein, the terms “a” or “an” are used, as is common in patentdocuments, to include one or more than one, independent of any otherinstances or usages of “at least one” or “one or more.”

As used herein, the term “or” is used to refer to a nonexclusive or,such that “A or B” includes “A but not B,” “B but not A,” and “A and B,”unless otherwise indicated.

As used herein, the term “about” is used to refer to an amount that isapproximately, nearly, almost, or in the vicinity of being equal to oris equal to a stated amount, e.g., the state amount plus/minus about 5%,about 4%, about 3%, about 2% or about 1%.

As used herein, the terms “Caenorhabditis elegans” or “C. elegans” referto a free-living transparent nematode, about 1 mm in length, which livein temperate soil environments. The basic anatomy of C. elegans includesa mouth, pharynx, intestine, gonad, and collagenous cuticle.

As used herein, the terms “electropharyngeogram” and “EPG” refer to anelectrophysiological recording of the pumping activity of the pharynx ofan organism (such as a nematode, for example, C. elegans). The EPG canbe recorded non-invasively with surface electrodes. The waveform of theEPG approximates the derivative of the action potential waveform andincludes the E or excitation phase (depolarization of the basalmembranes of the pharyngeal muscle cells), the P or plateau phase(membrane potential remains depolarized and muscle contraction occurs),and the R or repolarization phase (return of membrane potential tonegative values, muscle relaxation). The E phase includes two closelyspaced positive spikes (the corpus and the terminal bulb contractions)and the R phase includes two negative spikes corresponding to relaxationof the corpus and the terminal bulb, respectively. As describedthroughout the instant specification an EPG recording may not only beobtained for an individual organism but a composite EPG recording may beobtained from a pool of multiple nematodes compacted, for examplesurface to surface, between two or more electrodes within a recordingchannel of a microfluidic device. In embodiments, a composite EPGrecording may be obtained from a densely packed pool of multiplenematodes arranged between two or more electrodes within a fluid-filledrecording channel such that the nematodes occupied at least half thevolume of the recording channel between the electrodes.

As used herein, the term “fluidic device” refers to a device thatutilizes the flow of fluid to distribute substances and/or organisms(such as substances dissolved in a fluid and/or substances or organismssuspended in a fluid). A fluidic device can be of any dimension, as longas its dimensions are suitable to accommodate the size of substances ororganisms included or suspended in the fluid. In embodiments, a deviceis a microfluidic device that exploits the properties of fluid flow thatarise at length scales in the sub-millimeter range. One such property islaminar flow. In some examples, a microfluidic device has a channel orchamber with at least one dimension of 300 microns or less. In otherexamples, two dimensions are 300 microns or less. Some microfluidicdevices are fabricated in glass whereas others are fabricated in abio-compatible silicone or thermoplastic polymer by replica molding. Thelatter are referred to as soft-lithography microfluidic devices. Theterm “microfluidic device” is sometimes used as a synonym for the moregeneral term “microfabricated device,” which refers to an object thatmay or may not exploit the properties of fluid flow at thesub-millimeter scale.

As used herein, the term “nematode” refers to an organism that is amember of the phylum Nematoda, commonly referred to as roundworms.Nematodes include free-living species (such as the soil nematode C.elegans) and parasitic species. Species parasitic on humans includeascarids, filarias, hookworms, pinworms, and whipworms. It is estimatedthat more than two billion people worldwide are infected with at leastone nematode species. Parasitic nematodes also infect companion animalsand livestock, including dogs and cats (e.g., Dirofilaria immitis;heartworm), pigs (Trichinella spiralis), and sheep (e.g., Haemonchuscontortus). There are also nematode species which are parasitic oninsects and plants.

As used herein, the term “differential amplifier” refers to a type ofelectronic amplifier that amplifies the difference between two inputvoltages but suppresses any voltage common to the two inputs.

As used herein, the term “voltage-clamp amplifier” refers to anamplifier that is used to apply a voltage across a cell or organismwhile measuring current through the cell or organism. The two-electrodevoltage-clamp mode utilizes both a voltage-recording electrode and acurrent-injecting electrode for the control of membrane voltage.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety for allpurposes. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

Microfluidic Device and System

Provided herein are microfluidic devices, systems, and methods of usefor measuring and recording a composite electropharyngeogram (EPG)signal from a pool of multiple nematodes. The disclosed devices allowsimultaneous data collection from multiple nematodes, such as 10 ormore, in the same recording channel.

In embodiments, a first microfluidic device (e.g. 100) comprises aninlet port and outlet port directly connected to a funnel shaped holdingreservoir, a single recording channel connected in series to the holdingreservoir and two or more integrated electrodes directly connected tothe recording channel. In other embodiments is provided a microfluidicsystem for measuring and recording a composite electropharyngeogram(EPG) signal from a pool of multiple nematodes, wherein the systemcomprises an inlet port and outlet port directly connected to a holdingreservoir; a single recording channel connected in series to the holdingreservoir, two or more integrated electrodes directly connected to therecording channel, and at least one differential amplifier or at leastone voltage-clamp amplifier, wherein the amplifier is connected to anoutput from the two or more integrated electrodes.

In embodiments, the recording channel is configured to hold a pool ofmultiple nematodes that are present in an aqueous buffer solution. Inembodiments, the recording channel is configured to hold 10 to 10,000nematodes. The pool of multiple nematodes is placed through the inletport, which is wide enough to accommodate means of transferring anaqueous solution containing the pool of multiple nematodes into thereservoir. In certain embodiments, a syringe or pipette may be used totransfer an aqueous buffer containing the pool of multiple nematodesinto the reservoir. In embodiments, the recording channel is configuredto accommodate nematodes in various orientations. In certainembodiments, the pool of multiple nematodes is retained in the recordingchannel by positive pressure.

During use, the recording channel contains an electrically conductivebuffer solution (such as a saline solution) which provides electricalcontinuity between electrodes and the nematodes. In embodiments, thebuffer solution further comprises a stimulant that causes or inducespharyngeal pumping. In certain embodiments, the buffer comprisesserotonin or nematode food.

In embodiments, the pool of multiple nematodes is packed in between theat least two electrodes that are directly contacted to the recordingchannel. In embodiments, the recording channel is configured to hold 10to 10,000 nematodes between the at least two electrodes. In certainembodiments, the microfluidic device comprises a third electrode that isa ground electrode to reduce electrical noise.

In embodiments, electrical contact with the recording channel isachieved by means of electrodes embedded in the material that forms themicrofluidic device (such as an integrated electrode). Integratedelectrodes can be included in any suitable material (for example, glass,PDMS, polycarbonate, acrylic, or other polymeric material). Integratedelectrodes can be fabricated by any means that yields spatiallypatterned conductive elements that serve as wires. In one non-limitingexample, the electrodes are composed of indium tin oxide. In anotherexample, electrodes are composed of metallic silver. Patterning ofelectrode materials can be achieved for example, using photolithographycombined with etching.

In embodiments, a second microfluidic device (e.g., 500) comprises aninlet port and outlet port directly connected by a single fluidicfeature, a recording channel located between the inlet and outlet portsmolded into the bottom of a block that forms the microfluidic chip,which attached to a backing slip form the microfluidic device, and twoelectrodes that also function as the inlet and outlet port tube. Incertain embodiments, the electrodes are integrated electrodes. Incertain other embodiments, metal tubes placed in the inlet port andoutlet port function as electrodes. Those tubes, in certain embodimentsmay comprise a mesh over the end placed in the inlet or outlet portwhich functions to maintain the nematodes in the recording channel andallows for packing a high density of nematodes in the recording channel.In certain embodiments, a first tube comprising a mesh cover the end isplaced the outlet port before nematodes are loaded into the recordingchannel and a second tube optionally comprising a mesh covering the endis placed in the inlet port after the pool of nematodes are loaded andpacked in the recording channel.

In other embodiments is provided a microfluidic system for measuring andrecording a composite electropharyngeogram (EPG) signal from a pool ofmultiple nematodes, wherein the system comprises an inlet port andoutlet port directly connected to a recording channel, two integratedelectrodes directly connected to the recording channel, at least onedifferential amplifier, wherein the amplifier is connected to an outputfrom the two integrated electrodes fitted with clips and amplifiercables.

In embodiments, the recording channel of the second microfluidic deviceis configured to hold a pool of multiple nematodes that are present inan aqueous buffer solution. In embodiments, the pool of multiplenematodes is packed in between the two electrodes within the recordingchannel. In embodiments, the recording channel is configured to hold 10to 10,000 nematodes. The pool of multiple nematodes is placed throughthe inlet port, which is wide enough to accommodate means oftransferring an aqueous solution containing the pool of multiplenematodes, into the recording channel. The inlet and outlet portsinclude a tube-shaped electrode which in the case of the outlet port iscovered with a mesh to prevent the nematodes form exiting the recordingchannel while allowing a buffer solution to pass through the electrode.In certain embodiments, a syringe or pipette may be used to transfer anaqueous buffer containing the pool of multiple nematodes into therecording channel. In embodiments, the recording channel is configuredto accommodate nematodes in various orientations. In certainembodiments, the pool of multiple nematodes is retained in the recordingchannel by positive pressure.

In embodiments, electrical contact with the recording channel isachieved by means of electrodes embedded in the material that forms themicrofluidic device (such as an integrated electrode). The electrodes ofthe second microfluidic device have a dual function. The electrodesfabricated as a hollow tube made of a conducting material function as anelectrode and as the lining of the ports for both the inlet and outletport. In one non-limiting example, the electrodes are composed ofstainless-steel. Integrated electrodes can be included in any suitablematerial used to form the microfluidic chip (for example, glass, PDMS,polycarbonate, acrylic, or other polymeric material).

In certain embodiments, the recording channel, of the first or secondmicrofluidic device, is 10 mm to 500 mm in length and 10 μm to 500 μm inwidth.

In embodiments, the length of the first or second microfluidic devicerecording channel is about 10 mm, about 15 mm, about 20 mm, about 35 mm,about 50 nm, about 75 mm, about 100 mm, about 125 mm, about 150 mm,about 200 mm, about 250 mm, about 300 mm, about 350 mm, about 400 mm,about 450 mm or about 500 mm. In certain embodiments, the length of therecording channel is greater than 500 mm. In certain other embodiments,the length of the recording channel is from about 15 mm to about 150 mm,from about 15 mm to about 125 mm, from about 15 mm to about 100 mm, fromabout 15 mm to about 90 mm, from about 15 mm to about 80 mm, from about15 mm to about 70 mm, from about 15 mm to about 60 mm, from about 15 mmto about 55 mm, from about 15 mm to about 50 mm, from about 15 mm toabout 45 mm, from about 15 mm to about 40 mm, from about 15 mm to about35 mm, from about 15 mm to about 30 mm, or about 15 mm to about 25 mm.In exemplary embodiments, the length of the recording channel is about17 mm.

In embodiments, the width of the first or second microfluidic devicerecording channel is about 10 μm, about 20 μm, about 30 μm, about 40 μm,about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm,about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm,about 300 μm, about 325 μm, about 350 μm, about 375 μm, about 400 μm,about 425 μm, about 450 μm, about 475 μm or about 500 μm. In certainembodiments, the width of recording channel is greater about 500 μm. Incertain other embodiments, the width of the recording channel is fromabout 20 μm to about 200 μm, from about 20 μm to about 175 μm, fromabout 20 μm to about 150 μm, from about 20 μm to about 125 μm, fromabout 20 μm to about 100 μm, from about 20 μm to about 90 μm, from about20 μm to about 80 μm, from about 20 μm to about 70 μm, from about 20 μmto about 60 μm, from about 20 μm to about 55 μm, from about 20 μm toabout 50 μm, from about 20 μm to about 45 μm, from about 20 μm to about40 μm or from about 20 μm to about 35 μm. In exemplary embodiments, thewidth of the recording channel is about 30 μm.

In certain embodiments, a microfluidic device is made from anelastomeric material such as a silicone polymer (for example,poly(dimethyl siloxane) (PDMS)). Suitable PDMS polymers include, but arenot limited to Sylgard® 182, Sylgard® 184, and Sylgard® 186 (DowCorning, Midland, Mich.). In one non-limiting example, the PDMS isSylgard® 184. Additional polymers that can be used to make the disclosedmicrofluidic chip include acrylic, polyurethane, polyamides,polyethelyene, polycarbonates, polyacetylenes and polydiacetylenes,polyphosphazenes, polysiloxanes, polyolefins, polyesters (such asthermoset polyester (TPE)), polyethers, poly(ether ketones),poly(alkaline oxides), poly(ethylene terephthalate), poly(methylmethacrylate), polyurethane methacrylate (PUMA), polystyrene,thiol-enes, fluoropolymers (for example, perfluoropolyethers), NorlandOptical Adhesive 81, and derivatives and block, random, radial, linear,or teleblock copolymers, cross-linkable materials such as proteinaceousmaterials and/or combinations of two or more thereof. Also suitable arepolymers formed from monomeric alkylacrylates, alkylmethacrylates,alpha-methylstyrene, vinyl chloride and other halogen-containingmonomers, maleic anhydride, acrylic acid, and acrylonitrile. Monomerscan be used alone, or mixtures of different monomers can be used to formhomopolymers and copolymers. See, e.g., U.S. Pat. No. 6,645,432;McDonald et al., Electrophoresis 21:27-30, 2000; Rolland et al., J. Am.Chem. Soc. 126:2322-2323, 2004; Carlborg et al., Lab Chip 11:3136-3147,2011; Sollier et al., Lab Chip 11:3752-3765, 2011. In some examples, thechannel of the device (such as a device made from PDMS) can be coatedwith a sol-gel. See Abate et al., Lab Chip 8:516-518, 2008, for example.In other embodiments, suitable materials for making the disclosedmicrofluidic device include polymeric films, photoresist, hydrogels, orthermoplastic polymers.

In certain embodiments, a present first or second microfluidic devicecomprises a silicone polymer, thermoplastic polymer, an acrylic polymer,or a polycarbonate polymer. In exemplary embodiments, a silicone polymercomprises polydimethylsiloxane (PDMS) elastomer. In certain otherembodiments, the thermoplastic polymers comprise poly(methylmethacrylate) (PMMA), polycarbonate (PC), polystyrene (PS), polyvinylchloride (PVC), polyimide (PI), olefin polymers, cyclic olefin copolymer(COC), cyclic olefin polymer (COP), or cyclic block copolymer (CBC). Inexemplary embodiments, the microfluidic device comprises PDMS.

Microfluidic devices can be fabricated by methods known to one ofordinary skill in the art. In some embodiments, the disclosed devicesare made by molding uncured polymer from a photoresist master usingstandard photolithographic methods (e.g., U.S. Pat. No. 6,645,432;Madou, Fundamentals of Microfabrication, CRC Press, Boca Raton, Fla.,1997). In other embodiments, the disclosed devices are made by chemicaletching, laser cutting, photopolymerization, lamination, embossing, orinjection molding. In the case of glass devices, the microfluidic devicecan for instance be fabricated by etching the various types of channelsinto a thin glass plate and bonding this plate to a second glass platethat serves as a flat substrate. One of ordinary skill in the art canselect an appropriate fabrication method based on the selected materialfor the device.

FIG. 1 is a top view of one embodiment of the first microfluidic device100. The device includes the microfluidic chip 105 and a backing 110,wherein the chip 105 includes an inlet port 125, an outlet port 130,each connected to a reservoir 115 that is connected in series to arecording channel 120. The electrodes, 135, 140 and 145, are directlyconnected to the recording channel. An aqueous buffer comprising thepool of multiple nematodes flows from the inlet port 125, into thereservoir 115 and the recording channel 120, wherein the electricalsignal from the pool of multiple nematodes is recorded for thosenematodes between electrodes 135 and 145; electrode 140 is a ground thatreduces electrical noise. To aid in movement of nematodes into therecording channel 120, in certain embodiments, the microfluidic device100 is subjected to a spin that forces buffer solution and nematodesinto the recording channel 120. See Table 1 and A for exemplarydimensions of the microfluidic device 100.

In embodiments, the microfluidic device comprises a range of dimensionmeasurements. No limitation on the length or width of recording channel120 is intended wherein the dimensions inform the dimension of thereservoir 115 and together the reservoir 115 and recording channel 120define the outer dimensions of the microfluidic chip 105 andmicrofluidic device.

TABLE A Range of dimension measurement for microfluidic device 100Feature Width (mm) Length (mm) Depth (mm) Inlet port (125) 1-2 (diam)1-2 (diam) Depth of chip Outlet port (130) 1-2 (diam) 1-2 (diam) Depthof chip Holding reservoir (115) 10-50 10-50 0.01-0.30 Recording channel(120) 0.01-1    5-500 0.01-0.30 Chip (PDMS block) (105) 20-60  25-160 1-10

Exemplary depth of chip 105 dimensions is about 5 mm. See FIG. 1 .

FIG. 2 is an image of an exemplary microfluidic system 200. The systemincludes the microfluidic device 100 loaded in a recording dock 205 andconnected to a differential amplifier 210. In certain embodiments, therecording dock is connected to a voltage-clamp amplifier.

FIGS. 5A-5C and 6 provide a view of the second microfluidic device 500and system 600. A microfluidic chip 505 bonded to a backing 510 containsa single fluidic feature, a recording channel 515, molded into thebottom of the chip. The channel is connected to an inlet port 520, andan outlet port 525. Two metal tubes 530 and 535 form inlet 520 andoutlet 525 connections, respectively. The opening of the tube 535 iscovered by a mesh 540. The stainless-steel tubes 530, 535 contact theaqueous solution in the channel such that they also served aselectrodes.

See Table 4 and A for exemplary dimensions of the microfluidic device500.

TABLE 4 Dimensions of the Exemplary Microfluidic Device 500 of FIG. 5Width (mm) Length (mm) Top view x- Top view y- Feature dimensiondimension Depth (mm) Inlet port (520) 1-2 (diam) 1-2 (diam) 5 Outletport (525) 1-2 (diam) 1-2 (diam) 5 Stainless-steel tubes 1-2 (diam) 1-2(diam) 12.5 (530, 535) Recording channel (515) 0.01-1    5-500 0.01-0.30Chip (PDMS block) (505) 20-60 25-160  1-10

FIG. 6 is an image of an exemplary microfluidic system 600. The systemincludes microfluidic device 500, differential amplifier 210, amplifiercable 605 fitted with clips 610 to connect the amplifier to themicrofluidic device 500.

Methods

Disclosed herein are methods for recording a compositeelectropharyngeogram (EPG) signal from a pool of multiple nematodes. Inembodiments, the method comprises introducing the pool of multiplenematodes into the holding reservoir through the inlet port of a presentmicrofluidic device, wherein the pool of multiple nematodes is presentin an aqueous buffer, moving the pool of multiple nematodes into thesingle recording channel, measuring electrophysiological signals fromthe pool of multiple nematodes and recording the electrophysiologicalsignals as a single composite EPG.

In some embodiments, the system includes the microfluidic device, two ormore electrodes, one or more amplifiers, which are connected to outputsfrom each electrode, an oscilloscope, which receives input from theamplifier, a data acquisition unit, which receives input from theamplifier; and a computer, which receives input from the dataacquisition unit. In some examples, the system also includes a means forregulating flow of solutions through the device (such as a pump, forexample, a syringe pump). One of ordinary skill in the art can utilizethe systems disclosed herein to measure composite EPG activity of a poolof multiple nematodes. In embodiments the pool of multiple nematodescomprises C. elegans. In other embodiments, the pool of nematodescomprises parasitic nematodes.

The nematodes are introduced to the device by any convenient means. Insome examples, the nematodes are introduced into the device bytransferring the pool of multiple nematodes to the inlet port (which ispre-loaded with an aqueous buffer) and applying gentle pressure (forexample, from a syringe) to move the nematodes into the reservoir andthe recording channel. In other embodiments, once loaded in thereservoir, the microfluidic device is subjected to centrifugal forces tomove the nematodes into the recording channel. In certain embodiments,the cuticle of the nematode is made more permeable to drugs and testcompounds by means of chemical treatments and/or genetic mutations. Inother embodiments, the ability of the nematode to capture and/or excreteforeign chemicals is compromised by genetic mutation of endogenous pumpsand other proteins.

In certain embodiments, when using the second microfluidic device,nematodes maintained in the recording channel allow for exchange or flowof buffer through the recording channel. The nematodes trapped in therecording channel by a mesh over the outlet port can be introduced to anew buffer solution by flushing, from inlet port to outlet port, with afresh first buffer solution or a second buffer solution. The first orsecond buffer solution may include a test compound(s). The exchange ofbuffer solution allows for the screening of test compounds by recordinga composite electropharyngeogram (EPG) signal from a pool of multiplenematodes.

Electrical recordings are made using standard techniques known to one ofordinary skill in the art. In some examples, EPGs are recorded by ACdifferential amplifiers connected to metal electrodes integrated intothe device. Signals are displayed on oscilloscopes and recorded forlater analysis using a data acquisition system connected to a computerrunning data acquisition software. Data analysis is performed offlineafter experiments. Raw EPG recordings can be filtered to remove slowdrift and high-frequency noise. Filtered recordings can be subjected toa conventional algorithm, such as the fast Fourier transform, forcomputing the power spectrum of the recording to identify the averagepumping frequency of the pool of nematodes. The power spectrum can becomputed as function of time or experimental treatments, includingdrugs, mutants, and toxic compounds.

Measuring and recording a composite electropharyngeogram (EPG) signalfrom a pool of multiple nematodes requires a pool of nematodes of asimilar size. To obtain nematodes of a consistent size that arecompatible with the dimensions of a recording channel, a large number ofage synchronized eggs were deposited in a culture plate containing foodand cultivated to yield a large population of young adult nematodes. Thesize of the nematodes can be controlled by selecting the time forincubation. In an exemplary embodiment the eggs were deposited in aculture plate containing food and cultivated at 20° C. for 3 days.

In alternative embodiments, methods using filters can also be used toobtain an age/size synchronized population of nematodes for use in thepresent methods. See US Patent Publ. No. 2019/0090458.

The nematodes are prepared for recording by suspending the nematodes ina buffer, preferably an M9 buffer solution (See Example 2 for bufferdetails). To stimulate pharyngeal pumping, the final resuspension bufferused “serotonin M9 buffer solution” which contained 10 mM serotonin inaddition to the constituents in Table 3.

In embodiments, the nematodes are injected into the first microfluidicdevice (100) inlet port (125) filling the reservoir. The microfluidicdevice (100) is then centrifuged to pack the nematodes into the channel(120). The microfluidic device is inserted into a recording dock 205thereby connecting it to a differential amplifier 210, which amplifiedthe voltage difference between electrodes 135 and 145 with the thirdelectrode 140 serving as a ground electrode to reduce electrical noise.See Example 2.

In embodiments, the nematodes are injected into the second microfluidicdevice (500) inlet port (520), suspended in M9 buffer without serotonin,until the channel is filled to capacity, the nematodes being preventedfrom exiting the channel via the outlet port by the mesh covering theoutlet port electrode. The M9 buffer solution is replaced with a secondbuffer solution, the serotonin containing buffer, by injecting thesecond buffer into the inlet port until about 10 times the volume of therecording channel exits the outlet port. The microfluidic deviceelectrodes are connected to a differential amplifier 210, using thecords (605) and clip (610) to record the composite EPG. See Example 4.

Disclosed herein are methods for screening test compounds by recording acomposite electropharyngeogram (EPG) signal from a pool of multiplenematodes. In embodiments, the method comprises contacting the pool ofmultiple nematodes with the test compound, measuring and recording acomposite EPG from the contacted pool of multiple nematodes, comparingthe recorded composite EPG to a control composite EPG, and determiningif the recorded composite EPG is altered as compared to the controlcomposite EPG, whereby test compounds are screened.

In embodiments, determining if the recorded composite EPG is alteredcomprises determining the power spectrum of the composite EPG, thefrequency of the peak power of the composite EPG, the amplitude of thecomposite EPG, waveform of the composite EPG, or a combination thereof.In embodiments, the test compound is selected from a drug, a drugcandidate, an industrial chemical, or an environmental pollutant. Incertain embodiments, the drug or drug candidate is selected from anorganic compound, an inorganic compound, a hormone, a growth factor, acytokine, a receptor, an antibody, an enzyme, a peptide, an aptamer or avaccine.

In some embodiments, the disclosed methods include screening foranthelmintic or antimicrobial compounds. In other embodiments, themethods include screening for compounds of use for treatingneuromuscular diseases (such as muscular dystrophies, for example,Duchenne muscular dystrophy), neurodegenerative diseases (such asAlzheimer disease, Parkinson disease, Huntington disease, ortauopathies), mitochondrial disorders, or substance abuse disorders.

Methods of screening for or identifying anthelmintic compounds includeintroducing nematodes (such as C. elegans) in a device disclosed herein,contacting the nematode with one or more test compounds, and recording acomposite EPG from the nematodes, as disclosed above. The composite EPGin the presence of the one or more test compounds is compared to acontrol (such as a composite EPG from the same or a different C. elegansin the absence of the test compounds) and the compound is identified asan anthelmintic or candidate anthelmintic if the composite EPG isaltered (for example, the size and/or frequency of the EPG, or a portionthereof is decreased) in the presence of the test compound as comparedto the control. In embodiments, the nematodes are contacted withserotonin or bacterial food prior to and/or concurrent with the testcompound to stimulate pharyngeal pumping.

In embodiments, the pool of nematodes comprises: C. elegans; parasiticnematodes; transgenic or variant nematodes; nematodes that express oneor more human genes; or, wild type nematodes.

Methods of screening for or identifying compounds of potential use fortreating disease, such as neurodegenerative disease (for example,Parkinson disease, Huntington disease, Alzheimer disease), neuromusculardisease (for example, spinal muscular atrophies or amyotrophic lateralsclerosis), and muscular degenerative disease (for example, musculardystrophies or sarcopenia) and/or inhibiting or reducing aging includeintroducing nematodes (such as C. elegans) in a device disclosed herein,contacting the pool of multiple nematodes with one or more testcompounds, and recording a composite EPG from the nematodes, asdisclosed above. In certain embodiments, such as diseases for which theC. elegans genome contains a gene that is orthologous to the human geneimplicated in the disease, a strain is created or obtained in which thatgene is mutated and is utilized in the screening methods. Inembodiments, a strain is created in which the human gene is expressed inC. elegans by transgenic techniques. Strains that are disease models canbe used in drug screens by searching for compounds that mitigate one ormore phenotypes in C. elegans. This mitigation can be the result ofeither chronic or acute exposure to a test compound. In one embodiment,recording a composite EPG signal from the pool of multiple nematodes isused to test for mitigation of disease phenotypes consisting ofalterations in the behavior, physiology, and/or other aspects of thepharynx. In one non-limiting example, the C. elegans model for spinalmuscular atrophy (SMA) exhibits reduced rates of pharyngeal pumping. Acandidate compound in a drug screen for SMA is identified by a reductionor reversal of the reduced pumping phenotype as compared to a controlgroup (such as untreated C. elegans). In other embodiments, histogramsof interspike intervals are used to assess effects of treatments onpumping. In the case of some C. elegans disease models, the presence orabsence of a pharyngeal phenotype is unknown. In these embodiments, themicrofluidic device is used to test for such a phenotype. If apharyngeal phenotype exists, then the model can be used as above toscreen for drugs. Some controls in drug screening experiments would beto apply the test compound to wild type nematodes with the expectationthat changes in the pharyngeal phenotype are absent, or in a directionopposite to the change seen in the disease model. For example, a drugeffective against SMA might have no effect on wild type nematodes, or itmight increase the rate of pharyngeal pumping.

C. elegans is well-established as a model in aging research. The devicesdisclosed herein provide a means of assessing or screening the effectsof treatments (for example, genetic alterations, pharmaceuticalcompounds, and/or environmental conditions) on the process, extent andmechanism of aging. The C. elegans pharynx exhibits a decline in pumpingrate with increasing age. In some examples, the microfluidic devices areused to quantify the effects of treatments on aging. This is done bygrowing and maintaining nematodes under conditions of chronic exposureto the treatment and sampling pumping rate throughout the aging processby monitoring pumping rate in a present microfluidic device. Pumping isstimulated by contact with serotonin or bacterial food. In someexamples, controls include nematodes of similar ages that were notexposed the treatment.

Many nematode species are parasites of plants causing an estimated $100billion of worldwide crop losses annually. These nematodes also transmitdamaging viruses to plants. Available control measures are very limited,with most plant nematicides withdrawn from the market because ofenvironmental concerns. Many species of plant nematodes have anelaborate feeding apparatus, including a sharp stylet that isrhythmically protruded and retracted to pierce plant cell walls and pumpfluids during feeding (Wyss, Feeding behavior of plant parasiticnematodes in “The Biology of Nematodes, D. L. Lee, editor, 2002, Taylorand Francis, London). When plant nematodes are contacted with serotonin,this feeding apparatus, which is homologous to the pharynx of non-plantnematodes, emits electrical impulses that can be monitored byconventional EPG recording methods (Rolfe and Perry, Nematology 3:31-34,2001). Many plant nematodes are of a size that is compatible with thepresent microfluidic devices. Thus, also disclosed herein are methods ofassessing or screening the effects of treatments (for example, geneticalterations, pharmaceutical or other compounds, and/or environmentalconditions) on plant nematodes utilizing the devices disclosed herein.In some examples, the present microfluidic devices are used to quantifythe effects of treatments on feeding in plant nematodes. Pumping isstimulated by contact with serotonin in the device. The nematodes areexposed to the treatment chronically or acutely. In some examples,pumping rate is measured. In other examples, histograms of interspikeintervals are used to assess effects of treatments on pumping. Controlsinclude nematodes of the same species and age that are not exposed tothe treatment, for instance.

After nematodes, the most abundant parasitic nematodes are digenetictrematodes, also known as flukes or flatworms. They parasitize a broadrange of vertebrates, including humans and domestic animals, leading todisease and economic losses. Whereas nematodes have a complete digestivesystem, with a mouth at one end and an anus at the other, a fluke'smouth leads to a blind sac. However, like nematodes, many flukes havewell-developed pharynges, which are used to ingest blood or tissue fromhosts. Many species of parasitic and free-living flukes have been thesubjects of intense biological inquiry in laboratory settings. As is thecase for C. elegans, studies of free-living species can inform researchon parasitic species. The muscular pharynx is typically richlyinnervated by neurons containing various neurotransmitters andneuromodulators. For example, dopamine, allatostatin, and octopaminereceptors are present in the neural plexus innervating the pharynx ofthe non-parasitic freshwater flatworm Schmidtea mediterranea. In someexamples, the EPG devices disclosed herein are used to quantify theeffects of drugs and other treatments on feeding in trematodes.Trematodes are introduced into an EPG array that has been modified byadjusting the size of the channels to accommodate them. Pharyngealactivity is stimulated by contacting the animals with an appropriateneurotransmitter. The nematodes are exposed to the treatment chronicallyor acutely. In some examples, pumping rate is measured. In otherexamples, histograms of interspike intervals are used to assess effectsof treatments on pumping. Controls involve trematodes of the samespecies and age that are not exposed to the treatment, for instance.

Also disclosed herein are methods of identifying compounds that aretoxic or have toxic effects on an organism. In some embodiments, themethods include screening compounds for inhibitors of the HERG channel(for example, potentially cardiotoxic compounds). In other embodiments,the methods include screening compounds for toxicity, for examplepotential environmental toxicity.

Inhibition of the HERG potassium channel can cause long QT syndrome andpotentially fatal ventricular arrhythmias. Several compounds have beenwithdrawn from late stage clinical trials as a result of cardiotoxicitydue to HERG channel inhibition and screening for long QT effects is nowmandatory for new drug candidates. Therefore, methods to identifypotential HERG channel inhibitors early in drug development caneliminate potentially unsafe compounds prior to significant investmentand can streamline development of compounds that do not exhibitcardiotoxicity.

In embodiments, methods of identifying compounds that inhibit the HERGchannel include introducing nematodes (such as C. elegans) in a devicedescribed herein, contacting the nematodes with one or more testcompounds, and recording an EPG from the pool of multiple nematodes, asdisclosed above. The composite EPG in the presence of the one or moretest compounds is compared to a control (such as a composite EPG fromthe same or a different C. elegans in the absence of the test compound)and the compound is identified as an inhibitor of HERG if the compositeEPG is altered (for example, inhibited) in the presence of the testcompound as compared to the control. In some examples, the composite EPGis inhibited (for example, the size and/or frequency of the EPG, or aportion thereof is decreased) in the presence of the test compound ascompared to the control. In embodiments, the frequency of actionpotentials within the composite EPG is increased or reduced indicating,respectively, facilitation or inhibition of the composite EPG. In someexamples, the nematodes are contacted with serotonin or bacterial foodprior to and/or concurrent with the test compound to stimulatepharyngeal pumping.

Methods of screening for or identifying toxic compounds includeintroducing a pool of multiple nematodes (such as C. elegans) in adevice disclosed herein, contacting the nematodes with one or more testcompounds, and recording a composite EPG from the nematodes, asdisclosed above. The composite EPG in the presence of the one or moretest compounds is compared to a control (such as a composite EPG fromthe same or a different C. elegans in the absence of the test compound)and the compound is identified as toxic or potentially toxic if thecomposite EPG is altered (for example, inhibited) in the presence of thetest compound as compared to the control. In some examples, theamplitude of action potentials or frequency of action potentials isdecreased in the presence of the test compound as compared to thecontrol. In some examples, the nematodes are contacted with serotonin orbacterial food prior to and/or concurrent with the test compound tostimulate pharyngeal pumping. In some examples, the compound is anenvironmental toxin (such as a heavy metal), pesticide, herbicide,industrial chemical, or naturally occurring compound of interest.Exemplary compounds include, but are not limited to, those listed in the1989 OSHA Toxic and Hazardous Substances List.

The test compounds used in the present invention include, but are notlimited to drugs, drug candidates, biologicals, food components, herb orplant components, proteins, peptides, oligonucleotides, DNA and RNA. Inembodiments, the test compound is a drug, a drug candidate, anindustrial chemical, an environmental pollutant, a pesticide, aninsecticide, a biological chemical, a vaccine preparation, a cytotoxicchemical, a mutagen, a hormone, an inhibitory compound, achemotherapeutic agent or a chemical. In certain embodiments, the drugor drug candidate is selected from the group consisting of an organiccompound, an inorganic compound, a hormone, a growth factor, a cytokine,a reception, an antibody, an enzyme, a peptide, an aptamer or a vaccine.The test compound can be either naturally-occurring or synthetic and canbe organic or inorganic. A person skilled in the art will recognize thatthe test compound can be added to the pool of multiple nematodes and/orthe microfluidic device in an appropriate solvent or buffer.

In embodiments, the test compound includes pharmacologically activedrugs or drug candidates and genetically active molecules. Testcompounds of interest include chemotherapeutic agents, anti-inflammatoryagents, hormones or hormone antagonists, ion channel modifiers, andneuroactive agents. Exemplary of pharmaceutical agents suitable for thisinvention are those described in “The Pharmacological Basis ofTherapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996),Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Drugs Acting on the Central NervousSystem; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions;Drugs Affecting Renal Function and Electrolyte Metabolism;Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; DrugsAffecting Uterine Motility; Chemotherapy of Parasitic Infections;Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases;Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs;Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology,all incorporated herein by reference. Also included are toxins, andbiological and chemical warfare agents, for example see Somani, S. M.(Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).

In embodiments, the test compound includes all of the classes ofmolecules disclosed herein and may further or separately comprisesamples of unknown content. While many samples will comprise compoundsin solution, solid samples that can be dissolved in a suitable solventmay also be assayed. Samples containing test compounds of interestinclude environmental samples, e.g., ground water, sea water, or miningwaste; biological samples, e.g., lysates prepared from crops or tissuesamples; manufacturing samples, e.g., time course during preparation ofpharmaceuticals; as well as libraries of compounds prepared foranalysis; and the like. Samples of interest include test compounds beingassessed for potential therapeutic value, e.g., drug candidates fromplant or fungal cells.

Test compounds are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, naturally orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how touse the embodiments provided herein and are not intended to limit thescope of the disclosure nor are they intended to represent that theExamples below are all of the experiments or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. dimensions, amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, dimensions are in millimeters (mm), and temperatureis in degrees Centigrade. It should be understood that variations in themethods as described can be made without changing the fundamentalaspects that the Examples are meant to illustrate.

Example 1: Design and Fabrication of the Microfluidic Device (100)

Provided herein is a microfluidic device and system for measuring andrecording a composite electropharyngeogram (EPG) signal from a pool ofmultiple nematodes. See FIGS. 1 and 2 .

Provided herein is the design and fabrication of the microfluidic device100 and system 200 represented in FIGS. 1 and 2 . The microfluidicdevice was prepared with a rectangular block (e.g. microfluidic chip)105 composed of polydimethylsiloxane (PDMS) measuring 45 mm×14 mm×5 mmbonded to a 1 mm thick glass microscope slide (e.g. backing) 110measuring 75 mm×25 mm. Two fluidic features, connected in series, weremolded into the bottom of the block: a triangular shaped reservoir 115which accepts a population of, or a pool of, multiple nematodessuspended in a buffer solution, and a 0.30 mm wide recording channel120. The reservoir 115 had 1.5 mm diameter inlet port 125 and 1.5 mmdiameter outlet port 130. Three electrodes 135, 140 and 145, madecontact with any buffer solution filling the recording channel 120.Regions of the electrodes beneath the PDMS block 105 had a high lengthto width ratio to increase the fluidic resistance of these potentialleakage pathways.

The microfluidic device 100 was fabricated using single-layerphoto-lithography and single-layer soft-lithography techniques. Aphotomask containing the microfluidic features 115 (reservoir) and 120(recording channel) was drawn using Vectorworks 2017 CAD software andprinted on a transparency with a resolution of 25,400 dots per inch(CAD/Art Services, Bandon, Oreg.). A 3-inch silicon wafer was spincoated with SU8-2075 photoresist (Microchem Corp.) at 1740 revolutionsper minute (RPM) to obtain a height of 100 microns. The coated wafer wasexposed to UV light using the photomask. The wafer was developed andsilanized by treatment with(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane vapor (Gelest,Inc) in a vacuum chamber at 20° C. to reduce surface adhesion.Polydimethylsiloxane (PDMS, Dow Corning) was mixed in the ratio 10:1,degassed, and poured to a depth of 5 mm onto the silanized wafercontained in a 9 cm petri dish. The PDMS was cured for 3 hours at 65°C., excised, peeled off the wafer, and trimmed to size. Inlet port 125and outlet port 130 were formed using a 1.5 mm diameter punch. Forbonding, the PDMS block 105 (microfluidic chip) and glass slide 110(backing) were cleaned and activated by placing both parts in an airplasma (medium field strength, PDC-32G plasma cleaner, Harrick Plasma,Inc., USA) for 1 minute and immediately bringing the glass slide intocontact with the feature side of the PDMS microfluidic chip 105. Carewas taken to ensure that the recording channel contacted all threeelectrodes on the glass slide. Curing of the bond was accelerated bybaking the device for 30 minutes at 100° C. See FIG. 1 .

TABLE 1 Dimensions of Exemplary Microfluidic Device of FIG. 1 Width (mm)Length (mm) Top view x- Top view y- Feature dimension dimension Depth(mm) Inlet port (125) 1.5 (diam) 1.5 (diam) 5 Outlet port (130) 1.5(diam) 1.5 (diam) 5 Holding reservoir (115) 11 19.1 0.1 Recordingchannel (120) 0.3 17.5 0.1 Chip (PDMS block) (105) 14 45 5

The overall dimensions of the exemplary microfluidic device of FIG. 1include a width of 25 mm, a length of 75 mm and a depth of 6 mm. Inembodiments provided herein is a microfluidic device for measuring acomposite electropharyngeogram (EPG) signal from a pool of multiplenematodes comprising exemplary microfluidic chip 105. In embodiments, anexemplary microfluidic chip comprises an inlet port 125 and outlet port130 directly connected to a holding reservoir 115; a single recordingchannel 120, configured to hold 10 to 10,000 nematodes, connected inseries to the holding reservoir 115; and, two or more integratedelectrodes 135 and 145 directly connected to the recording channel. Incertain embodiments, the microfluidic chip 105 is attached to a backing110 to form an exemplary microfluidic device.

Provided herein is an exemplary microfluidic system 200 for measuring acomposite electropharyngeogram (EPG) signal from a pool of multiplenematodes comprising exemplary microfluidic device 100 and a recordingdock 205 and differential amplifier 210. In certain embodiments, thesystem further comprises a frame that holds the microfluidic device 100,which is then placed in the recording dock 205.

Example 2: Methods of Measuring and Recording a CompositeElectropharyngeogram (EPG) Signal from a Pool of Multiple NematodesUsing the Device of Example 1

Provided herein is a method for measuring and recording a compositeelectropharyngeogram (EPG) signal from a pool of multiple nematodesusing the microfluidic device 200 of FIG. 2 featuring a microfluidicdevice 100, a recording dock 205, and a differential amplifier 210.

To obtain nematodes of a consistent size that were compatible with thedimensions of the recording channel 120, we utilized a common procedureto obtain a large number of age synchronized eggs. This involveddissolving gravid hermaphrodites in a bleaching solution the ingredientsof which are shown in Table 2. Eggs were deposited in a culture platecontaining food and cultivated at 20 C for 3 days, yielding a largepopulation of young adult nematodes.

TABLE 2 Bleaching Solution Constituent Amount (mL) Distilled water 3.675Sodium hypochlorite solution (5%) 1.200 NaOH solution (10M) 0.125

To prepare nematodes for recording, 2 mL of “normal M9 buffer solution,”defined in Table 3, was added to a culture plate containing young adultnematodes grown from synchronized eggs. The plate was swirled to liftnematodes into the solution and the contents were poured into a 2 mLEppendorf tube. Nematodes were cleaned of adherent bacteria and debrisby 5 cleaning cycles each of which involved pelleting nematodes in acentrifuge (1.2 g, 30 sec), drawing off the supernatant, andresuspending in M9 buffer. To stimulate pharyngeal pumping, the finalresuspension used “serotonin M9 buffer solution” which contained 10 mMserotonin in addition to the constituents in Table 3.

TABLE 3 M9 Buffer Solution Constituent Amount KH₂PO₄ 3 g Na₂HPO₄ 6 gNaCl 5 g MgSO₄ (1M) 1 mL Distilled water to 1 L

For filling the microfluidic device, a 5 mL syringe was fitted with a 50cm length of polyethylene tubing (1.4 mm ID, 1.9 mm OD), with astainless-steel tube (1.2 mm ID, 1.47 mm OD, 12.7 mm long) inserted halfway into the open end of the tubing to facilitate connection with theinlet port of the device. The syringe and tubing were filled withserotonin M9 buffer which was injected into the inlet port 125 untilsolution began to escape from the outlet port 130 and filling thereservoir 115. For transferring nematodes into the microfluidic device,approximately 10 uL of fluid from the pellet of nematodes in anEppendorf tube was drawn into the tube of a second syringe, prepared inthe same manner as the first syringe, then injected into the inlet port125. The microfluidic device was placed in a 50 mL centrifuge tube andspun for at 420 g for 5 min, packing nematodes into in the recordingchannel 120. This procedure yielded approximately 25 nematodes betweenelectrodes 135 and 145. The microfluidic device was inserted into arecording dock 205 thereby connecting it to a differential amplifier 210(ScreenChip System, NemaMetrix, Inc.) which amplified the voltagedifference between electrodes 135 and 145. The third electrode 140served as a ground electrode to reduce electrical noise.

According to circuit theory, (i) the waveform of a composite EPGrecorded at electrodes 135 and 145 should be the sum of the voltagedifferences generated by each nematode at its unique location andorientation with respect to the electrodes and other nematodes, and (ii)the peak frequency of the waveform's power spectrum should represent theaverage pumping frequency of the population of nematodes between theelectrodes. We validated this theory by recording single-nematode EPGsfrom 22 nematodes in serotonin M9 buffer solution for 10-14 min using acommercially available recording system (ScreenChip System, NemaMetrix,Inc.). See U.S. Pat. No. 9,723,817. Segments of three of the individual22 nematode EPG recordings are shown in FIG. 3A-C. The average pumpingfrequency of the 22 nematodes was 4.9±0.13 Hz (SEM). FIG. 3D shows aportion of the synthetic (in silico) composite EPG recording which wascomputed by taking the sum of all 22 single-nematode EPG measurements.This recording shows periods of synchronous, high amplitude activity(indicated by horizontal lines) alternating with periods ofasynchronous, low amplitude activity. This is the waveform envelopeexpected for a population of oscillators operating at similar but uniquefrequencies such that they periodically come in and out of phase, aphenomenon commonly referred to as “beating”. The power spectrum of thisrecording had a peak frequency of 5.1 Hz (FIG. 3E), which agrees wellwith the average pumping frequency of the 22 single-nematode EPGrecordings.

FIG. 4A shows a multi-nematode (composite) EPG recorded in serotonin M9buffer solution from a pool of multiple nematodes utilizing microfluidicsystem 200. Two lines of evidence indicate that the recorded activityrepresents pharyngeal pumping. First, the envelope of this tracestrongly resembles the envelope of the synthetic composite EPG recordingin FIG. 3D in that it also exhibits periods of synchronous, highamplitude activity (indicated by horizontal lines) alternating withperiods of asynchronous, low amplitude activity. No such envelope wasobserved in the voltage trace recorded in normal M9 buffer withoutserotonin to stimulate pharyngeal pumping (FIG. 4B), a condition inwhich there should be little or no pumping. Second, the power spectrumof recorded activity had a prominent peak at 5.6 Hz (FIG. 4C), inagreement with the pumping frequency of single nematodes exposed to thesaturating concentration of serotonin used in this experiment, and thefact that this peak was absent in power spectrum of the voltage tracerecorded in normal M9 buffer solution.

Example 3: Design and Fabrication of Microfluidic Device (500)

Provided herein is a microfluidic device and system for measuring andrecording a composite electropharyngeogram (EPG) signal from a pool ofmultiple nematodes. See FIGS. 5 and 6 .

Provided herein is the design and fabrication of the microfluidic device500 and system 600 represented in FIGS. 5 and 6 . The microfluidicdevice was prepared with a rectangular block (e.g. microfluidic chip)505 composed of polydimethylsiloxane (PDMS) measuring 45mm×14 mm×5 mmbonded to a 1 mm thick glass microscope slide (e.g. backing) 510measuring 75 mm×25 mm. A single fluidic feature was molded into thebottom of the block: a recording channel 515 measuring 90 μm high, 300μm wide, and 20 mm long. The channel was connected to a 1.5 mm diameterinlet port 520, and 1.5 mm diameter outlet port 525. Two stainless-steeltubes 530 and 535 measuring, 1.5 mm (diameter)×12.5 mm were insertedinto the PDMS block 505 to form inlet 520 and outlet 525 connections,respectively. The opening of the stainless-steel tube 535 within thePDMS was covered by a nylon mesh 540 having square pores measuring 11 μmon a side. The channel was filled with an aqueous buffer solution. Thestainless-steel tubes 530, 535 contacted the aqueous solution such thatthey also served as electrodes.

The microfluidic device 500 was fabricated in the same manner as thedevice 100 except that the 3-inch silicon wafer was spin coated withSU8-2075 photoresist (Microchem Corp.) at 2559 revolutions per minute(RPM) to obtain a height of 90 μm.

TABLE 4 Dimensions of the Exemplary Microfluidic Device 500 of FIG. 5Width (mm) Length (mm) Top view x- Top view y- Feature dimensiondimension Depth (mm) Inlet port (520) 1-2 (diam) 1-2 (diam) 5 Outletport (525) 1-2 (diam) 1-2 (diam) 5 Stainless-steel tubes 1-2 (diam) 1-2(diam) 12.5 (530, 535) Recording channel (515) 0.01-1    5-500 0.01-0.30Chip (PDMS block) (505) 20-60 25-160  1-10

The overall dimensions of the exemplary microfluidic device of FIG. 5comprises a width of 25 mm, a length of 75 mm and a depth of 6 mm. Inembodiments provided herein is a microfluidic device for measuring acomposite electropharyngeogram (EPG) signal from a pool of multiplenematodes comprising exemplary microfluidic chip 505. In embodiments, anexemplary microfluidic chip comprises an inlet port 520 and outlet port525 directly connected to a single recording channel 515, configured tohold 10 to 10,000 nematodes and two or more stainless-steel tubes 530and 535 directly connected to the recording channel via the inlet andoutlet port 520, 525. In certain embodiments, the microfluidic chip 505is attached to a backing 510 to form an exemplary microfluidic device.

Provided herein is an exemplary microfluidic system 600 of FIG. 6 formeasuring a composite electropharyngeogram (EPG) signal from a pool ofmultiple nematodes comprising exemplary microfluidic device 500,differential amplifier 210, amplifier cable 605 fitted with clips 610 toconnect the amplifier to the microfluidic device 500, and a platform 615which anchors the cable 605 and clips 610, and has a hole 620 in thecenter so the device 500 can be viewed under transillumination on aconventional stereomicroscope.

Example 4: Methods of Measuring and Recording a CompositeElectropharyngeogram (EPG) Signal from a Pool of Multiple NematodesUsing the Device of Example 3

Provided herein is a method for measuring and recording a compositeelectropharyngeogram (EPG) signal from a pool of multiple nematodesusing the microfluidic system 600 of FIG. 6 featuring a microfluidicdevice 500, differential amplifier 210, amplifier cable 605 fitted withclips 610, for example micro test lead hooks, to connect the amplifierto the microfluidic device 500.

To obtain nematodes of a consistent size that were compatible with thedimensions of the recording channel 515, we used the method of Example2. Alternative methods using filters can also be used to obtain anage/size synchronized population of nematodes for use. See US PatentPubl. No. 2019/0090458.

To prepare nematodes for recording, we used the method of Example 2,except that the final resuspension used “normal M9 buffer solution,”defined in Table 3.

For filling the microfluidic device, we used the method of Example 2,except that the syringe and tubing were filled with “normal M9 buffersolution.”

FIG. 7A shows a typical segment of a multi-nematode (composite) EPGrecorded for 16 minutes in Solution A, normal M9 buffer solution, from apool of multiple nematodes utilizing microfluidic system 600. At the endof this recording, the normal M9 buffer solution was replaced withSolution B, serotonin M9 buffer solution disclosed in Example 1, withoutdislodging the original pool of nematodes. Replacement was accomplishedby exchanging the syringe and tubing filled with normal M9 buffersolution for a syringe and tubing filled with serotonin M9 buffersolution. Gentle pressure was applied to the syringe until approximately5 μL of fluid flowed out of the outlet port, corresponding toapproximately 10 times the volume of the recording channel. The nylonmesh 540 covering the opening of the stainless-steel tube 535 within thePDMS block 505 prevented the nematodes from leaving the recordingchannel while the fluid was exchanged.

FIG. 7B shows a typical segment of a multi-nematode (composite) EPGrecorded for 25 minutes in Solution B from the same pool of multiplenematodes recorded in FIG. 7A, again utilizing the microfluidic system600. FIG. 7C shows the power spectrum of the recordings obtained inSolution A and Solution B, together with a third trace showing thedifference between the first two traces. The difference trace revealed aprominent peak centered at 5 Hz, which indicates the specific effect ofserotonin on the power spectrum. The center frequency of this peak iswithin the expected range of pharyngeal pumping frequency. This result,like the result illustrated in FIG. 4C, confirms that the recordedactivity corresponds to pharyngeal function. However, as the samepopulation of nematodes was recorded under conditions of the twodifferent buffer solutions in FIG. 7C, but not in FIG. 4C, FIG. 7Cestablishes, in addition, that the system 600 can be used to measure theeffect of drugs or other test compounds on pharyngeal pumping. Thisfinding is significant because it establishes the feasibility of an “Aversus B” experimental design, a widely used screening approach in whichthe effects of the drug in Solution B are obtained by comparison with aninternal control, here solution A, applied to the same pool or set oftest organisms.

Importantly, the use of the A versus B experimental design is notlimited to measuring the effect of drugs like serotonin, which directlyinduces pharyngeal pumping. This design can also be used to measure theeffect of a drug or other test compound that modulates pharyngealpumping frequency only after it has already been initiated. For example,this could be done by measuring the difference in average pumpingfrequency in solutions A and B, where solution A is serotonin M9 buffersolution, and solution B is serotonin M9 buffer solution plus a testcompound such as an anthelmintic. In another example, this could be doneby subtracting the power spectra of the recordings obtained in solutionsA and B where, again, where solution A is serotonin M9 buffer solution,and solution B is serotonin M9 buffer solution plus a test compound suchas an anthelmintic.

1-77. (canceled)
 78. A microfluidic device for measuring a composite electropharyngeogram (EPG) signal from a pool of multiple nematodes comprising: a) an inlet port and outlet port directly connected to a single recording channel; b) the single recording channel, configured to hold 10 to 10,000 nematodes; c) a filter integrated into the recording channel to retain nematodes in the single recording channel; and, d) two or more electrodes connected to the recording channel.
 79. The device of claim 78, wherein the recording channel is 10 mm to 500 mm in length and 10 μm to 500 μm in width.
 80. The device of claim 78, wherein the recording channel is about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 350 μm, about 375 μm, about 400 μm, about 425 μm, about 450 μm, about 475 μm or about 500 μm in width.
 81. The device of claim 78, wherein the device comprises a silicone polymer, a thermoplastic polymer, an acrylic polymer, or a polycarbonate polymer.
 82. The device of claim 81, wherein the silicone polymer comprises a polydimethylsiloxane (PDMS) elastomer.
 83. The device of claim 78, wherein the device comprises a backing.
 84. A microfluidic system for recording a composite electropharyngeogram (EPG) signal from a pool of multiple nematodes comprising: a) an inlet port and outlet port directly connected to a single recording channel; b) a single recording channel, configured to hold 10 to 10,000 nematodes; c) a filter integrated into the recording channel to retain nematodes in the single recording channel; d) two or more electrodes connected to the recording channel; and, e) at least one differential amplifier or at least one voltage-clamp amplifier, wherein the amplifier is connected to an output from the two or more electrodes.
 85. The system of claim 84, wherein the differential amplifier or voltage-clamp amplifier is connected to the two or more integrated electrodes via a recording dock.
 86. The system of claim 84, wherein the recording channel is 10 mm to 500 mm in length and 10 μm to 500 μm in width.
 87. The system of claim 84, wherein the recording channel is about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 350 μm, about 375 μm, about 400 μm, about 425 μm, about 450 μm, about 475 μm or about 500 μm in width.
 88. The system of claim 78, comprising a silicone polymer, a thermoplastic polymer, an acrylic polymer, or a polycarbonate polymer.
 89. The system of claim 88, wherein the silicone polymer comprises a polydimethylsiloxane (PDMS) elastomer.
 90. A method for recording a composite electropharyngeogram (EPG) signal from a pool of multiple nematodes comprising: a) introducing the pool of multiple nematodes into the single recording channel through the inlet port of the microfluidic system of claim 84, wherein the pool of multiple nematodes is present in an aqueous buffer solution; b) measuring electrophysiological signals from the pool of multiple nematodes; and, c) recording the electrophysiological signals as a single composite EPG.
 91. The method of claim 90, wherein the buffer solution comprises serotonin, food, or other stimulant to cause pharyngeal pumping.
 92. The method of claim 90, wherein the pool of nematodes comprises transgenic nematodes or transgenic nematodes comprising a gene variant.
 93. The method of claim 90, wherein the pool of multiple nematodes is perfused with a second buffer solution.
 94. The method of claim 93, wherein the recording step c) is repeated following the perfusion with the second buffer.
 95. A method for screening test compounds by recording a composite electropharyngeogram (EPG) signal from a pool of multiple nematodes using the microfluidic system of claim 84, comprising: a) contacting the pool of multiple nematodes with the test compound; b) measuring and recording a composite EPG from the contacted pool of multiple nematodes; c) comparing the recorded composite EPG to a control composite EPG; and, d) determining if the recorded composite EPG is altered as compared to the control composite EPG, whereby test compounds are screened.
 96. The method of claim 95, wherein determining if the recorded composite EPG is altered comprises determining the power spectrum of the composite EPG, the frequency of the peak power of the composite EPG, the amplitude of the composite EPG, waveform of the composite EPG, or a combination thereof.
 97. The method of claim 95, wherein the test compound is selected from a drug, a drug candidate, an industrial chemical, or an environmental pollutant. 